To better understand the role of GCK and GCKR in vivo, the murine Gck and Gckr genes have been isolated. Both Gck-/- and Gckr -/- mice have been created and backcrossed on to a C57Bl/6 background used to generate double knock-out (KO) mice. The mutations did not affect mouse development as the Gck, Gckr, and double KO mice are born with normal Mendelian frequencies. We have nearly completed the analysis of these mice and are now focused on the double KO mice as they exhibit a sharp reduction in the number of follicular B cells, an expansion of marginal zone B cells, hypergammaglobulemia, defective humoral responses to neo-antigens, and evidence of autoimmunity. B lymphocyte recirculation through lymph nodes requires crossing endothelial barriers and chemoattractant-triggered cell migration. We have shown how lymph node anatomy and chemoattractant receptor signaling organize B lymphocyte trafficking through lymph nodes. Surprisingly B and T cells differ in their entrances and initial trafficking through high endothelial venules and their entrance into lymph node follicle and T cell zone, respectively. We have also begun a study of how local immunization alters homeostatic lymphocyte trafficking through the draining lymph node. We have shown how immunization induced changes in lymph node architecture along with cell intrinsic factors facilitate and how these changes help coordinate the trafficking of B cells into and through lymph nodes following immunization. We have shown that immunization leads to a rapid recruitment of newly arrived B cells into the lymph node follicle and causes the cells to be retained within the follicle. We are currently trying to understand the underlying mechanisms that alters B lymphocyte trafficking. We have also begun to examine how various antigens are delivered to B and T cells in the lymph node by intravital microscopy. In addition we have developed methods for intravital imaging lymphoid and hematopoietic progenitor in the bone marrow. As a potential downstream effector in the chemokine receptor signaling pathway, we have examined the functional role of the non-muscle myosin Myo1e, which is significantly enriched in B lymphocytes. We have established Myo1e-/- mice and are studying the consequences of the loss of this protein on B lymphocyte function. We have also crossed the Myo1e-/- mice with mice lacking Myo1f, another non-muscle myosin that is enriched in both B and T lymphocytes. In addition, we have been studying chemoattractant receptor signaling in mice lacking Gnai2 and Gnai3 in their B cells. This results in a profound loss of chemoattractant receptor function and a severe phenotype. Autophagy delivers cytoplasmic constituents to autophagolysosomes and is linked to both innate andadaptive immunity. Toll-like receptor 4 (TLR4) signaling induces autophagy and recruits Beclin-1, the mammalian homolog of yeast Atg6, to the receptor complex. We found that tumor necrosis factor receptor (TNFR)associated factor 6 (TRAF6)mediated, Lys63 (K63)linked ubiquitination of Beclin-1 is critical for TLR4-triggered autophagy in macrophages. Inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1 beta and IL-18. Suggesting interplay between these pathways, lipopolysaccharide induces inflammasome activation in macrophages genetically deficient in autophagy proteins, but not in wild type macrophages. We have shown that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered RalB activation and autophagosome formation. The induction of autophagy did not depend upon ASC or capase-1, but did depend upon the inflammasome sensor. Blocking autophagy potentiated inflammasome activity while stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes. Severe acute respiratory syndrome (SARS) is a recently recognized viral infectious disease. A 2002-3 international outbreak occurred where patients suffered a severe respiratory illness with close to 10% mortality. Recent reports of SARS-like illnesses portend possible new outbreaks. Here, we identify three mechanisms by which open reading frame-9b (ORF-9b) of the SARS coronavirus (SARS-CoV) contributes to disease pathogenesis. ORF-9b localizes to mitochondria causing mitochondrial elongation by ubiquitination and proteasomal degradation of DRP1, a host protein involved in mitochondrial fission; it inhibits antiviral immune responses by targeting the mitochondrial-associated adaptor molecule MAVS signalosome for PCBP2/AIP4 dependent destruction; and it triggers ATG5-dependent autophagy. Our data indicate that SARS-CoV ORF-9b manipulates host cell mitochondrial function to help evade host innate immunity. In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Galpha i3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and formation of LC3 puncta in bone marrow derived macrophages from wild type, Gnai3-/-, Gpsm1-/-, or Rgs19-/- mice following amino acid starvation or nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3-/-, and Gpsm1-/- macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Galpha i nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Galpha i3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Galpha i signaling may impact autophagy in some cell types it does not in macrophages.